It is well known that certain proteinaceous compounds have the ability to substitute in a highly effective manner for sugar in giving foods and beverages a sweet taste. The simplest of these examples is aspartame, which is a dipeptide derivative and currently on the market. However, two much more complex proteins, monellin and thaumatin have been isolated from plant sources. Thaumatin is isolated from Thaumatococcus daniellii, a West African plant having triangular shaped fruit at ground level. The natural protein product, thaumatin, has an average sweetness of 2500 times that of sucrose and has been marketed under the trademark Talin. The three-dimensional structure of this protein has been studied and the results published by De Vos, A. M., et al., Proc Natl Acad Sci USA (1985) 82:1406-1409.
The other protein is isolated from "Serendipity Berries" of the West African Plant Dioscoreophyllum comminisii. The amino acid sequence of monellin is known, and the three-dimensional structure of this protein has been determined by Ogata, C., et al., Nature (1987) 328:739-742. Monellin has been characterized by Morris et al., J Biol Chem (1973) 248:534-539, and by others; Cagan, Science (1973) 181:32-35; Bohak and Li, Biochim Biophys Acta (1976) 427:153-170; Hudson and Beeman, Biochem Biophys Res Comm (1976) 71:212-220; Van der Wel and Loeve, FEBS Lett (1973) 29:181-183; Frank and Zuber Hoppe-Seyler's Z Physiol Chem (1976) 357:585-592; Morris and Cagan, Biochimn Biophys Acta (1972) 261:114-122. U.S. Pat. No. 3,998,798 describes the preparation of natural monellin.
The known amino acid sequence of the A and B chains of natural monellin is shown in FIG. 1. It is a two chain peptide, one "A" chain containing 45, and the other "B" chain, 50 amino acid residues. The three-dimensional conformation of the protein, shown in FIG. 2, is evidently essential for its activity because when native monellin is heated to 90.degree. C. at neutral pH or to 50.degree. C. at acidic pH and then cooled, the sweetness is destroyed.
The three-dimensional conformation of the protein as recently determined (supra) is shown in FIG. 2. A B chain containing 50 amino acids is intimately associated with the A chain of 45 amino acids in such a way that there are many interchain interactions. Heating of the protein, evidently dissociates the chains in such a way that they are incapable of reforming into the appropriate conformation.
It has now been found that the conformation of this proteinaceous compound can be maintained by synthesizing portions of the B and A components on a single molecule. This constraint results in resistance to denaturation and ease of renaturation and of maintaining the three-dimensional conformation. Furthermore, the proteinaceous compound can be made by synthetic or recombinant techniques, and the uncertainties and expense of extraction from natural sources are thereby obviated.